Assay of free and complexed prostate-specific antigen

ABSTRACT

According to the method of the invention, immunoassays are applied to measure free PSA as well as a proteinase inhibitor complex. Free PSA and PSA complex are according to the invention measured by a non-competitive immunoassay employing at least two different antibodies. The invention is further characterized in that the PSA proteinase inhibitor complex of interest is formed with α 1  -antichymotrypsin. Moreover, the invention is characterized in that free PSA, the PSA-proteinase inhibitor complex and their ratio are applied in the diagnosis of patients with prostate cancer.

The present invention relates to an immunoassay of prostate-specificantigen (PSA), in which specific reagent materials (antibodies) are usedthat allow the measurement of free PSA as well as the PSA proteinaseinhibitor complex.

It also relates to the use of free PSA and the PSA proteinase inhibitorcomplex and their ratio as a useful marker in diagnosis of patients withprostate cancer.

BACKGROUND OF THE INVENTION

The prostate specific antigen (PSA) was first purified from prostatictissue (Wang et al. Invest Urol 1979), but the same protein was almostsimultaneously and independently characterized in the seminal plasma(Hara et al. J Lab Clin Med 1989; Graves et al. N Engl J Med 1985). PSAis now known to be a 33-kDa glycosylated single chain serine protease(Lilja, J Clin Invest 1985; Watt et al. Proc Natl Acad Sci (USA) 1986).The 237 amino-acid polypeptide backbone has extensive similarities withthat of the glandular kallikreins (Lundwall et al. FEBS Lett 1987;Schaller et al. Eur J Biochem 1987). Unlike the trypsin-like glandularkallikreins, which display Arg-restricted substrate specifity (MacDonaldet al. Biochem J 1988), PSA displays chymotrypsin-like substratespecificity (Akiyama et al. FEBS Lett 1987; Christensson et al.Manuscript 1990; Lilja et al. J Biol Chem 1989). PSA has been predictedto be produced as a presumably inactive zymogen (Lundwall et al. FEBSLett 1987). Active PSA is secreted into the seminal plasma (Lilja, JClin Invest 1985) where it is one of the most abundant proteins of theprostate (Lilja et al. The Prostate 1988; Dube/ et al. J Androl 1987).The biological activity of PSA in semen relates to its limitedproteolytic fragmentation of the predominant proteins secreted by theseminal vesicles (Lilja, J Clin Invest 1985; Lilja etal. J Clin Invest1987; McGee etal. Biol Reprod 1988).

Secondary to the release from the prostate epithelium PSA may also bedetected in the circulation (Papsidero etal. Cancer Res 1980).Measurements of the serum concentration of PSA have now found widespreaduse in monitoring of patients with prostate cancer, although increasedserum concentrations of PSA have also been reported in benign prostatichyperplasia and secondary to surgical trauma of the prostate (Duffy, AnnClin Biochem 1989; Brawer et al. Urology suppl 1989). However, it ispresently unknown whether the immunoreactivity in serum represents thePSA-zymogen, the active PSA or PSA inactivated by extracellularproteinase inhibitors and contradictory results have been reported onthe molecular mass of this immunoreactivity. Papsidero reported in 1980the PSA-immunoreactivity to elute as a single 90 to 100 kDa peak(Papsidero etal. Cancer Res 1980), whereas Alfthan and Stenman reportedthe predominant part of this immunoreactivity to elute as a 30-kDaprotein (Alfthan etal. Coin Chem 1988) when subjected to gel filtrationchromatography.

In the proceeding invention we showed that PSA has the ability to formcomplexes with proteinase inhibitors that occur in high concentration inthe human extracellular fluids and that PSA occurs in these fluids bothin a free and complexed form. In addition, the invention proved to bevery useful in diagnosis of prostate cancer patients.

SUMMARY OF THE INVENTION

According to the method of the invention, immunoassays are applied tomeasure free PSA as well as PSA as a proteinase inhibitor complex. FreePSA and the PSA complex are according to the invention measured by anon-competitive immunoassay employing at least two different monoclonalantibodies. The invention is further characterized in that the PSAproteinase inhibitor complex of interest is formed either with α₂-antichymotrypsin, α₁ -protease inhibitor (API) or α₂ -macroglobulin.Moreover, the invention is characterized by that free PSA, thePSA-proteinase inhibitor complex and their ratio are applied in thediagnosis of patients with prostate cancer.

DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a polyclonal antibody and three monoclonal antibodiesused to probe proteins blotted onto PVDF-membranes after agarose gelelectrophoresis. In lane 1 is 1 μg PSA, in lane 2 is 1 μg PSA incubatedat 37° C. for 30 min with 6 μg of α₁ -antichymotrypsin, and in lane 3 is6 μg of α₁ -antichymotrypsin.

FIG. 2 presents a polyclonal and three monoclonal antibodies used toprobe proteins blotted onto PVDF-membranes after SDS-PAGE. In lane 1 is1 μg PSA, in lane 2 is 1 μg PSA incubated at 37° C. for 30 min with 6 μgof α₁ -antichymotrypsin, and in lane 3 is 6 μg of α₁ -antichymotrypsin.

FIG. 3 presents the specificity of the three assay versions.

FIG. 4 presents a correlation of PSA immunoreactivity in serum samplesfrom 65 individual patients when analyzed with assay versions A and C.

FIG. 5 presents a correlation of PSA immunoreactivity in serum samplesfrom 65 individual patients when analyzed with assay versions A and B.

FIG. 6 presents a gel filtration of a patient sample B on a TSK 250 HPLCcolumn. The PSA immunoreactivity of the eluted fractions were analyzedby assay versions A, B and C.

FIG. 7 presents a gel filtration of a patient sample C on a TSK 250 HPLCcolumn. The PSA immunoreactivity of the eluted fractions were analyzedby assay versions A, B and C.

FIG. 8 presents a gel filtration of a patient sample D on a TSK 250 HPLCcolumn. The PSA immunoreactivity of the eluted fractions was analyzed byassay versions A, B and C.

FIG. 9 presents a characterization of PSA immunoreactivity in a serumsample with a PSA level of 10000 μg/l by gel filtration. PSA and thePSA-ACT complex were measured by IFMA.

FIG. 10 presents the proportion of the PSA-ACT complex of the total PSAimmunoreactivity in sera of patients with prostatic cancer as a functionof the PSA concentration. The level of PSA was measured by IRMA and thatof PSA-ACT by IFMA.

FIG. 11 presents the concentration of the PSA-API complex measured byIFMA as a function of the PSA concentration measured by IRMA in serafrom patients with prostatic cancer.

DETAILED DESCRIPTION 1. Production and characterization of monoclonalantibodies

Production of anti-PSA specific monoclonal antibodies Balb/c mice wereimmunized by intraperitoneal injections with 70 μg of PSA emulsified inequal volumes with Freund's complete adjuvant. The immunization wasrepeated after 3, 6 and 9 weeks with 50 μg of PSA emulsified withFreund's incomplete adjuvant. Three weeks later the mice were given afinal booster with 40 μg of PSA and the mice were killed four dayslater. Lymphoid cells of the spleen were prepared and mixed in a 1:1ratio with plasmacytoma cells (NS-1). The cells were fused and harvestedin microtiter wells in KC-2000 (Hazleton Biologics Inc., Le nexa, USA)containing 200 g/L foetal calf serum and HAT-supplement H-0262 (1:50,Sigma) (Matikainen et al. J Gen Microbiol 1983).

Anti-PSA specific antibody production by the master clones was assayedwith well strip plates coated with rabbit antimouse IgG (Lo/ vgren etal. Talanta 1984). The strips were incubated with either the hybridomasupernatants or the standard (monoclonal antibody against PSA; 0812Hybritech), washed, incubated with Eu-labelled PSA (50 ng per well), andthe amount of bound Eu-labelled PSA was determined.

Cloning of the master clones by limited dilutions was performed asdescribed (Staszewski and Yale, J Biol Med 1984). The desired cloneswere expanded intraperitoneally in Balb/c mice; the ascitic fluid beingcollected in 10 days. The IgG-fraction of the ascitic fluid was purifiedby chromatography on protein A-Sepharose following the protocolrecommended by the manufacturer.

Solid phase bound PSA was used to test if one unlabelled monoclonalantibody could block the binding of another Eu-labelled anti-PSAMAb tothe solid-phase bound PSA. The solid-phase bound PSA was obtained by theincubation of 25 μl aliquots of purified PSA (25 μg/L) and 200 μL ofAssay-buffer DELFIA^(R) (50 mmol/L Tris, pH 7.75, 0.15 mol/L NaCl, 0.5g/L BSA, and 0.5 g/L NAN₃) for 2 h in well strip plates coated with the2E9 or the 5A10 anti-PSAMAb. The strips were washed and then incubatedfor 1 h with 200 μL of one unlabelled anti-PSA MAb (0.005-50 μg/L).Again, the strips were washed, incubated for 1 h with anotherEu-labelled anti-PSAMAb and the amount of bound Eu-labelled anti-PSA wasdetermined.

Partial characterization of the epitope specificity of three monoclonalantibodies against PSA

Several clones produced monoclonal antibodies against PSA as indicatedby fluorometric assay. Three of these (designated 2E9, 2H11 and 5A10)(The cell line designated 5Al was deposited with the EuropeanCollection-of Animal Cell Cultures, Public Health Laboratory ServiceCentre for Applied Microbiology & Research, Porton Down, Salisbury,Wilts. SP4 OJG, U.K., on 12 Mar. 1993 and assigned accession number93031201. The cell line is identified as 5A10E7F11H4.) were expanded andthe antibodies isolated from the ascitic fluid. The three monoclonalantibodies against PSA were used to probe proteins blotted ontoPVDF-membranes after agarose gel electrophoresis (FIG. 1) or SDS-PAGE(FIG. 2) of 1 μg of PSA (lane 1); 1 μg of PSA incubated at 37° C. for 30min with 6 μg of α₁ -antichymotrypsin (lane 2); and 6 μg of α₁-antichymotrypsin (lane 3). PSA blotted to PVDF-membranes from theagarose gels was identified by all three monoclonal antibodies whereasthe FSA complexed to α₁ -antichymctrypsin was identified by the 2E9 andthe 2H11 antibodies but not by the 5A10 antibody. The 2E9 antibody wasthe only anti-PSA Mab that readily identified PSA and PSA compiexed toα₁ -antichymotrypsin when these proteins were blotted ontoPVDF-membranes after SDS-PAGE. However, a minute reaction was alsoobtained with the PSA (but not with the PSA complexed to α₁-antichymotrypsin) when the 2H11 and the 5A10 antibodies were used toprobe these proteins blotted onto PVDF-membranes after SDS-PAGE.

The epitope specificity of the three monoclonal antibodies was alsocharacterized using three different sets of solid-phase sandwich assays.Thereby assay (A), where the 2E9 antibody was used as solid-phasecatcher and Eu-labelled 2E11 was used as detecting antibody, displayedan almost identical dose-response for PSA as compared with PSA complexedto α₁ -antichymotrypsin (Table 1; FIG. 3). This contrasts with bothassay (B), where the 5A10 antibody was used as catcher and Eu-labelled2H11 was used as detecting antibody, which preferentially recognized PSAbut only poorly recognized PSA complexed to α₁ -antichymotrypsin, andwith assay (C), where the 2E9 antibody was used as catcher andEu-labelled antibody against α₁ -antichymotrypsin was used as detectingantibody, which only recognized PSA complexed to α₁ -antichymotrypsin(Table 1; FIG. 3).

Solid-phase bound PSA was used to further characterize the epitopespecificity of the three monoclonal antibodies against PSA; thesolid-phase binding of PSA having been achieved by the use of well stripplates coated with the 2E9 or the 5A10 antibody. It was thereby foundthat none of the anti-PSAMAb's 2E9, 2H11 or the 5A10 significantlyblocked the binding of each other when we tested the ability of oneanti-PSAMAb to block the binding of another Eu-labelled anti-PSAMAb tothe solid-phase bound PSA.

2. The occurrence of PSA-proteinase inhibitor complexes in human serumAnalysis of PSA in human serum

Sera from individual patient samples (n=65) were analyzed with the threedifferent sets of assays (A, B and C). Regression analysis of theresults obtained with assay A and assay C gave y=0.89x+6.55, r=0.97(FIG. 4); and the regression analysis between assay A and assay B gavey=0.10x+9.56, r=0.82 (FIG. 5).

The total recovery of the immunoreactivity from the gel filtrationexperiments of patient samples on the TSK 250 HPLC column was equallyhigh (82 to 107%) with all three assay procedures used (A, B and C). Thegel filtration experiments of patient samples on the TSK 250 HPLC columnshowed that the predominant peak of PSA-immunoreactivity, when analyzedwith assay A, was identified in fractions eluting at a positioncorresponding to a molecular mass of 80 to 90 kDa while a minor peak ofthis immunoreactivity was found in fractions eluting at a positioncorresponding to a molecular mass of 25 to 40 kDa (FIG. 6-8). In muchthe same way, the analysis of the fractions eluted with assay C(specific for PSA complexed to α₁ -antichymotrypsin) identified onepredominant immunoreactive peak in the range 80 to 90 kDa (FIG. 6-8).However, when assay B was used to analyze the fractions eluted from thegel filtration experiments the predominant immunoreactive peak eluted ata position corresponding to a mass of 25 to 40 kDa. The elution positionof this peak corresponded to the minor immunoreactive peak identifiedwith assay A (FIG. 6-8).

When serum samples from men with various levels of PSA (10-10,000 μg/L)were fractionated by gel filtration, two components corresponding to PSAand PSA-ACT were also observed. In samples with high PSA-levels thePSA-ACT complex dominated (FIG. 9). In female sere these components werenot seen (not shown). The proportion of PSA-ACT of total PSAimmunoreactivity increased with increasing PSA levels (FIG. 10). in serefrom health./males with PSA levels below 2.8 ug/L the proportion ofPSA-ACT was 23-47%, in samples with PSA levels of 2.8-10 μg/L theproportion was 26-86 % and in samples with higher levels the proportionincreased further being 70-100 % at PSA levels over 1000 μg/L (FIG. 10).

On the basis of =he concentrations of the complexes expressed inarbitrary units the main complex of PSA in sere was PSA-ACT complex. Insamples with low PSA levels the concentration of both PSA-API andPSA-ACT were close to the detection limit. Therefore it was not possibleto calculate the proportion of these complexes in normal samples.Clearly elevated levels of PSA-API complex occurred in samples with PSAlevels over 40 μg/L and the levels tended to increase with increasinglevels of PSA (FIG. 11).

3. PSA and PSA-α₁ -antichymotrypsin complexes in the diagnosis ofpatients with prostate cancer

The three assay versions referred to under the section "Characterizationof the epitope specificity of three monoclonal antibodies against PSA"were used to test 144 patients with benign prostatic hyperplasia (BPH)and 122 patients with different stages of prostate cancer (CAP). Theratios between A: PSA complexed with α₁ -antichymotrypsin/PSA total andB: PSA free noncomplexed/PSA total were calculated as well as theclinical sensitivity and specificity for the measurement of total PSAand PSA α₁ -antichymotrypsin alone (Table 2). It is obvious from thepresented data that increased clinical specificity is achieved bymeasuring the PSA α₁ -antichymotrypsin complex and that the ratiosbetween PSA free/PSA total and PSA free/PSA complexed with α₁-antichymotrypsin are significantly different between BPH and CAPpatients.

Table 1

Table 1 presents a dose-response of purified PSA and PSA complexed to α₁-antichymotrypsin when analyzed by three different sets of assays.

The assay A is 2E9 anti-PSA MAb as solid phase catcher and Eu-labelled2H11 anti-PSA MAb as detecting antibody.

The assay B is 5A10 anti-PSAMAb as solid phase catcher and Eu-labelled2H11 anti-PSAMAb as detecting antibody.

The assay C is 2E9 anti-PSAMAb as solid phase catcher and Eu-labelledrabbit antibody against α₁ -antichymotrypsin as detecting antibody.

The column 1 indicate the purified PSA and columns 2 indicate the PSAcomplexed to α₁ -antichymotrypsin.

Tables 2a and 2b

Tables 2a and 2b present the results of the testing of the patientsamples with three assay versions for free, complexed and total PSA. Inthe table BPH indicates benign prostatic hyperplasia, CAP indicatesprostate cancer, G indicates the differentiation grade and T indicatesthe grade. Table 2b presents the sensitivity and the specifity.

                                      TABLE 1    __________________________________________________________________________    PSA assay    PSA A           B           C    μg/L        1     2     1     2     1    2    __________________________________________________________________________     1   6664  5250  6733 2119  435   4208     5  26897 23535 31487 3179  487   15662     10 53452 41064 65146 4573  559   30283    100 534860              460464                    600057                          33006 2105 267223    500 2231640              1826790                    2631640                          156712                                12073                                     726596    __________________________________________________________________________     1. Purified PSA; 2. PSA complexed to α.sub.1 -antichymotrypsin.

                  TABLE 2a    ______________________________________                                  Ra-                          Correlation                                  tio                          coefficient                                  mean    ______________________________________    BPH (n = 144) A. PSA c/PSA tot                                0.932     0.970                  B. PSA f/PSA tot                                0.853     0.302    CAP (n = 122) A.            0.994     1.219                  B.            0.784     0.191    CAP, G1 (n = 31)                  A.            0.994     1.628                  B.            0.922     0.190    CAP, G2 (n = 47)                  A.            0.972     1.141                  B.            0.956     0.169    CAP, G3 (n = 43)                  A.            0.996     1.014                  B.            0.818     0.218    CAP T1-2 (n = 56)                  A.            0.985     1.044                  B.            0.868     0.178    CAP T3-4 (n = 65)                  A.            0.993     1.372                  B.            0.770     0.204    CAP T4 (n = 25)                  A.            0.997     1.174    (not treated) B.            0.825     0.188    BPH (n = 84) PSA ≦ 5                  A.            0.879     1.059                  B.            0.850     0.301    BPH (n = 60) PSA > 5                  A.            0.888     0.846                  B.            0.735     0.303    CAP (n = 26) PEA ≦ 5                  A.            0.913     1.773                  B.            0.826     0.232    CAP (n = 94) PSA > 5                  A.            0.993     1.063                  B.            0.778     0.188    CAP (n = 25)  A.            0.919     1.025    PSA >  5 ≦ 20                  B.            0.502     0.187    CAP (n = 69)  A.            0.993     1.080    PSA > 20      B.            0.770     0.184    ______________________________________

                  TABLE 2b.    ______________________________________    Sensitivity and specificity    ______________________________________    PSA tot Sensitivity                       PSA tot   >5   95/121 = 0.785                                 >10  80/121 = 0.661            Specificity                       PSA tot   <5   84/144 = 0.583                                 <10  116/144 = 0.806    PSA c   Sensitivity                       PSA c     ≧5                                      93/121 = 0.769                                 >10  81/121 = 0.669            Specificity                       PSA c     <5   92/144 = 0.639                                 <10  124/144 = 0.861    ______________________________________

I claim:
 1. An immunoassay of prostate-specific antigen (PSA) characterized in thatthe fraction of prostate-specific antigen in complex with α₁ -antichymotrypsin is measured using one or more antibodies.
 2. An immunoassay according to claim 1 characterized in thatthe fraction of prostate-specific antigen in complex with α₁ -antichymotrypsin is measured by a non-competitive immunoassay.
 3. An immunoassay according to claim 2 characterized in thatthe fraction of prostate-specific antigen in complex with α₁ -antichymotrypsin is measured by a non-competitive immunoassay employing at least two different antibodies.
 4. An immunoassay according to claim 3 characterized in that the antibodies used for the recognition of the PSA-α₁ -antichymotrypsin complex bind either tothe PSA-α₁ -antichymotrypsin complex or to α₁ -antichymotrypsin.
 5. An immunoassay method for determining the amount of free noncomplexed prostate specific antigens in a sample comprising the steps of exposing the sample to antibodies specific to the free non-complexed prostate-specific antigens, and detecting the amount of antibodies which are bound to the free non-complexed prostate-specific antigens.
 6. An immunoassay according to claim 5 characterized in that the amount of free non-complexed prostate-specific antigen is measured by a non-competitive immunoassay.
 7. An immunoassay according to claim 6 characterized in that the amount of free non-complexed prostate-specific antigen is measured by a non-competitive immunoassay employing at least two different antibodies.
 8. An immunoassay according to claim 7 characterized in that the antibodies used for recognition of free non-complexed prostate-specific antigen (free PSA) bind toprostate specific antigen, and that at least one of the epitopes defined by the antibodies is not exposed (available) when the prostate specific antigen is in complex with α₁ -antichymotrypsin.
 9. An immunoassay of prostate-specific antigen (PSA) characterized in that the ratio between a) the fraction of free non-complexed prostate-specific antigen and b) the fraction of prostate-specific antigen in complex with α₁ -antichymotrypsin is measured using one or more antibodies that makes it possible to discriminate between free and complexed PSA.
 10. An immunoassay according to claim 9 characterized in that the ratio between a) the fraction of free non-complexed prostate-specific antigen and b) the fraction of the prostate-specific antigen in complex with α₁ -antichymotrypsin is measured by a non-competitive immunoassay.
 11. An immunoassay according to claim 10 characterized in that the ratio between a) the fraction of free non-complexed prostate-specific antigen and b) the fraction of the prostate-specific antigen in complex with α₁ -antichymotrypsin is measured by a non-competitive immunoassay employing at least three different antibodies.
 12. An immunoassay according to claim 11 characterized in that the antibodies used for the recognition of the PSA-α₁ -antichymotrypsin complex bind either tothe PSA-α₁ -antichymotrypsin complex or to α₁ -antichymotrypsin.
 13. An immunoassay of prostate-specific antigen (PSA) characterized in that the ratio between a) the fraction of prostate-specific antigen complexed with α₁ -antichymotrypsin and b) immunologically recognized total prostate-specific antigen is measured.
 14. An immunoassay of prostate-specific antigen (PSA) characterized in that the ratio between a) the fraction of free non-complexed prostate-specific antigen and b) immunologically recognized total prostate-specific antigen is measured.
 15. A method for differentiation between benign prostatic hyperplasia and prostate cancer by determining the ratio between free PSA: immunologically recognized total PSA in a patient's serum.
 16. A method for screening prostate cancer by determining the ratio between free PSA: immunologically recognized total PSA in a patient's serum.
 17. A method for differentiation between benign prostatic hyperplasia and prostate cancer by determining the ratio between free PSA:PSA complexed with α₁ -antichymotrypsin in a patient's serum.
 18. A method for screening prostate cancer by determining the ratio between free PSA:PSA complexed with α₁ -antichymotrypsin in a patient's serum.
 19. A method for differentiation between benign prostatic hyperplasia and prostate cancer by determining the ratio between PSA complexed with α₁ -antichymotrypsin: immunologically recognized total PSA in a patient's serum.
 20. A method for screening prostate cancer by determining the ratio between PSA complexed with α₁ -antichymotrypsin: immunologically recognized total PSA in a patient's serum. 